1. Kinesin-5 Is Essential for Growth-Cone Turning
Vidya C. Nadar, Andrea Ketschek, Kenneth A. Myers, Gianluca Gallo, Peter W. Baas 
Current Biology 
Volume 18, Issue 24, Pages (December 2008)
DOI: /j.cub
Copyright © 2008 Elsevier Ltd Terms and Conditions

2. Figure 1
Inhibition of Kinesin-5 Inhibits Growth-Cone Turning at a Laminin-Polylysine Border, Inhibits Turning toward NGF-Coated Beads, and Prevents Reorganization of MTs in Response to NGF-Coated Beads
(A and B) Axons (green, tubulin) grow on laminin (red), and their growth cones reach the border. (A) Growth cones of control explants treated with 0.1% DMSO turn at the border. (B) In the presence of 100 μM monastrol, most growth cones cross the border. Scale bar represents 15 μm.
(C) Quantification of the ratio of MT fluorescence on polylysine (axons that crossed the border) and laminin (axons that turned at the border) shows a significant increase in cultures treated with monastrol (red bar) compared to DMSO (blue bar). Asterisk denotes p ≤ Data represented as mean ± SEM.
(D) Quantification of the ratio of MT fluorescence, as above, shows that when kinesin-5 was depleted with the use of siRNA, a result similar to that obtained with monastrol treatment was observed. Asterisk denotes p ≤ Data represented as mean + SEM.
(E and F) Time-lapse phase-contrast images of growth cones of chicken DRG explants treated with DMSO (control) or monastrol for inhibition of kinesin-5. (E) Example of a growth cone, treated with 0.1% DMSO, turning toward the NGF-coated bead. (F) Example of a growth cone, treated with 100 μM monastrol, that had contacted the NGF-coated bead but did not turn toward the bead. Scale bar represents 10 μm.
(G) Percentage of growth cones that turned toward the NGF-coated bead. Asterisk denotes p ≤
(H and I) Examples of MT (green) and actin (red) organization in a DMSO-treated growth cone (H and H′) and a monastrol-treated growth cone (I and I′) contacting an NGF-coated bead. The axonal axis (see Supplemental Experimental Procedures) is depicted as a blue line. Bead locations are shown as dotted circles denoting the circumference of the beads. The control growth cone shows an increased MT distribution toward the contact point with the bead, but the monastrol-treated growth cone does not show any marked asymmetry in MT distribution.
(J) Quantification of the MDR (as defined in Supplemental Experimental Procedures) in control and monastrol-treated growth cones. The blue bar indicates the MDR in control growth cones that contact the bead. The red bar indicates the monastrol-treated growth cones that contact the bead. The yellow and green bars indicate growth cones that do not contact beads in the cases of control and monastrol-treated growth cones, respectively. Asterisk denotes p < Scale bar represents 10 μm. Data represented as mean + SEM.
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3. Figure 2
MT Behaviors in Growth Cones after Experimental Manipulations of Kinesin-5 and Cytoplasmic Dynein
(A–D) Examples of fluorescence micrographs. (A) Neuron transfected with control siRNA and treated with DMSO. (B) Neuron transfected with DHC siRNA and treated with DMSO. (C) Neuron transfected with control siRNA and treated with monastrol. (D) Neuron transfected with DHC siRNA and treated with monastrol. Scale bar represents 5 μm.
(E) Graph showing the number of EB3 comets that enter the filopodia in growth cones. Single asterisk denotes p ≤ 0.01; double asterisk denotes p ≤  (F) Graph showing the velocity of EB3 comets that enter the filopodia. Single asterisk denotes p ≤ 0.01; double asterisk denotes p ≤ (E and F) Red bars indicate neurons transfected with control siRNA and treated with DMSO. Blue bars indicate neurons simultaneously depleted of DHC and treated with DMSO. Yellow bars indicate neurons treated with the combination of control siRNA and monastrol, and green bars indicate neurons treated with the combination of DHC siRNA and monastrol. Data represented as mean + SEM.
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4. Figure 3 Distribution and Phosphorylation of Kinesin-5 in Growth Cones
(A and B) Fluorescence micrographs of growth cones stained with total kinesin-5 antibody (A) and phospho-kinesin-5 antibody (B). (A) Growth cone stained for total kinesin-5; (A′) the same growth cone, stained for MTs; and (A″) the overlay of A and A′. (B) Growth cone stained for phospho-kinesin-5; (B′) the same growth cone, stained for MTs; and (B″) the overlay of kinesin5 and MT. The box in (B′) is shown in higher magnification in “P.” Green arrowheads in “P” show the MTs in the P-domain, and the red region shows phospho-kinesin-5. The blue squiggly line indicates the perimeter of the P-domain. Scale bar represents 5 μm.
(C) Quantification of the polarized distribution of kinesin-5 in growth cones. The ratio of one side of kinesin-5 levels to the other is shown for growth cones immunostained with antibodies for total kinesin-5 (brown bar) and phospho-kinesin-5 (orange bar). Asterisk denotes p ≤ Data represented as mean + SEM.
(D) Quantification of the levels of kinesin-5 in the T-zone, C-domain, and axonal shaft. The values are ratioed against the levels of MTs in the same region. Brown bars indicate total kinesin-5, and the orange bar indicates phospho-kinesin-5. Asterisk denotes p ≤ Data represented as mean + SEM.
(E) Quantification of the levels of phospho-kinesin-5 and MTs on each half of the growth cones. Red bars indicate the levels of phospho-kinesin-5, and green bars indicate the levels of MTs on that side of the growth cone in the P-domain. Single asterisk denotes p ≤ 0.05; double asterisk denotes p ≤ Data represented as mean + SEM.
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5. Figure 4
Distribution of Phospho-Kinesin-5 in Growth Cones in the Border Assay
(A–C) Growth cones stained for MTs (green) and phospho-kinesin-5 (red) at different stages of the laminin (blue)-polylysine (white dotted line) border preparation. The leading edges of the growth cones are shown with a pink dotted line. A white arrow denotes the localization of phospho-kinesin-5. (A) Growth cone that has not encountered the border. (B) Growth cone that has contacted the border. (C) Growth cone that has made a turn at the border.
(D) Quantification of the percentage of growth cones that show localization of phospho-kinesin-5. A red bar indicates growth cones that have not reached the border, a green bar indicates growth cones that have contacted the border, and a blue bar indicates the growth cones that have already turned. Asterisk denotes p ≤ Scale bar represents 5 μm.
(E) Illustration of how forces exerted by different motor proteins may act during growth-cone turning. Arrows show the direction of the forces exerted by each motor. Dynein-based forces (red arrows) enable MTs to enter into the P-domain by opposing myosin-II-based forces (blue arrowheads). Kinesin-5 (purple and pink) is enriched in the T-zone, but its phosphorylation state may exhibit asymmetry across the growth cone (purple, high phosphorylation; pink, low phosphorylation). It becomes more phosphorylated on the side opposite the direction of turning (purple). When phosphorylated, kinesin-5 is able to generate forces (purple with blue arrows) in the T-zone that oppose the dynein-based forces. As a result, the MTs preferentially invade the P-domain on the side of the growth cone where kinesin-5 is less phosphorylated (pink). This scenario is consistent with our previously proposed model, in which the forces that influence the distribution of long MTs in growth cones are generated by the same motor proteins that regulate the transport of short MTs within the axonal shaft [21].
Current Biology  , DOI: ( /j.cub )
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